Legionella pneumophila (Lp) is the agent of Legionnaires' disease, a common and potentially fatal form of pneumonia. Lp is ubiquitous in natural and man-made water systems and infects the lungs after the inhalation of contaminated aerosols. In water and the lungs, Lp grows as an intracellular parasite, infecting either aquatic protozoa or alveolar macrophages. Previously, we demonstrated that Lp expresses a type II secretion (T2S) system that mediates the secretion of >25 proteins (substrates) and is required for optimal infection of both amoebal and mammalian host cells and the murine lung. During the last grant period, we documented that secreted PlaC (an acyltransferase), ProA (a metalloprotease), SrnA (a ribonuclease), NttA, and Lpw18401, and two other novel substrates promote intracellular growth/survival in Acanthamoeba, Hartmannella, and Naegleria amoebae in a unique host-specific fashion. We also demonstrated that T2S and a subset of novel substrates enhance intracellular growth/survival within lung macrophages and likely do so by helping to retain host protein Rab1 on the Legionella-containing vacuole. Finally, among other results, we documented that T2S and at least two of its substrates, i.e., ProA and NttA, dampen the chemokine and cytokine output of infected macrophages, a process that would most likely impede the clearance of Lp from the infected lung. This subversion of cytokine production was due, in part, to dampened signal transduction and cytokine gene transcription. In sum, we have been at the forefront of demonstrating that T2S is critical in all of the major facets of the Lp lifecycle and its associated disease, elaborating more substrates and encoding a wider variety of activities than is currently appreciated for any other bacterial T2S system. In the current proposal, we will now determine i) mechanisms by which PlaC, ProA, SrnA, and NttA and other novel T2S substrates enhance Lp growth in multiple amoebae, which are the essential links in Lp disease transmission from water systems to humans, ii) how T2S and its novel substrates Lpw02811 and Lpw18401 act to modulate Rab1 in macrophages, which are the critical host cell in the Lp-infected mammalian host, and iii) the mechanisms by which T2S and its substrates, including NttA, modulate cytokine signaling in Lp-infected macrophages as well as assess the impact of cytokine subversion on the cellular inflammatory response in the Lp-infected lung. The proposed studies will i) increase significantly our understanding of the pathogenesis of Lp, which is an important and increasing public health concern within the US and throughout the world, ii) expand our molecular understanding of both bacterial protein secretion and intracellular infection, iii) have implications for other important pathogens that utilize or are predicted to use T2S, iv) have implications for other bacteria that are pathogenic by virtue of being intracellular parasites of macrophages, and v) offer potential new targets for disease diagnosis, treatment, or prevention.